The present disclosure relates generally to the design of engineered organisms and, more specifically to organisms having selected genotypes for the production of muconic acid.
Terephthalate (also known as terephthalic acid and PTA) is the immediate precursor of polyethylene terephthalate (PET), used to make clothing, resins, plastic bottles and even as a poultry feed additive. Nearly all PTA is produced from para-xylene by the oxidation in air in a process known as the Mid Century Process (Roffia et al., Ind. Eng. Chem. Prod. Res. Dev. 23:629-634 (1984)). This oxidation is conducted at high temperature in an acetic acid solvent with a catalyst composed of cobalt and/or manganese salts. Para-xylene is derived from petrochemical sources, and is formed by high severity catalytic reforming of naphtha. Xylene is also obtained from the pyrolysis gasoline stream in a naphtha steam cracker and by toluene disproportion.
PTA, toluene and other aromatic precursors are naturally degraded by some bacteria. However, these degradation pathways typically involve monooxygenases that operate irreversibly in the degradative direction. Hence, biosynthetic pathways for PTA are severely limited by the properties of known enzymes to date.
Muconate (also known as muconic acid, MA) is an unsaturated dicarboxylic acid used as a raw material for resins, pharmaceuticals and agrochemicals. Muconate can be converted to adipic acid, a precursor of Nylon-6,6, via hydrogenation (Draths and Frost, J. Am. Chem. Soc. 116; 399-400 (1994)). Alternately, muconate can be hydrogenated using biometallic nanocatalysts (Thomas et al., Chem. Commun. 10:1126-1127 (2003)).
Muconate is a common degradation product of diverse aromatic compounds in microbes. Several biocatalytic strategies for making cis,cis-muconate have been developed. Engineered E. coli strains producing muconate from glucose via shikimate pathway enzymes have been developed in the Frost lab (U.S. Pat. No. 5,487,987 (1996); Niu et al., Biotechnol Prog., 18:201-211 (2002)). These strains are able to produce 36.8 g/L of cis, cis-muconate after 48 hours of culturing under fed-batch fermenter conditions (22% of the maximum theoretical yield from glucose). Muconate has also been produced biocatalytically from aromatic starting materials such as toluene, benzoic acid and catechol. Strains producing muconate from benzoate achieved titers of 13.5 g/L and productivity of 5.5 g/L/hr (Choi et al., J. Ferment. Bioeng. 84:70-76 (1997)). Muconate has also been generated from the effluents of a styrene monomer production plant (Wu et al., Enzyme and Microbiology Technology 35:598-604 (2004)).
All biocatalytic pathways identified to date proceed through enzymes in the shikimate pathway, or degradation enzymes from catechol. Consequently, they are limited to producing the cis, cis isomer of muconate, since these pathways involve ring-opening chemistry. The development of pathways for producing trans, trans-muconate and cis,trans-muconate would be useful because these isomers can serve as direct synthetic intermediates for producing renewable PTA via the inverse electron demand Diels-Alder reaction with acetylene. The present invention satisfies this need and provides related advantages as well.